The present invention relates generally to the field of viral vectors and specifically to a novel recombinant replication competent retrovirus useful for the transfer and expression of nucleic acid sequences in a targeted cell.
The development of genetic vectors has heralded the fast-growing field of somatic gene transfer. (Anderson, W. F., Science, 1984, 226:401-409). Vectors based on simple retroviruses, such as the Moloney Leukemia Virus (MoMLV), are often selected because they efficiently integrate into the genome of the target cell. Integration is thought to be a prerequisite for long-term expression of the transduced gene. However, efficient gene transfer to tumor tissue has been a major impediment to treatment of cell proliferative disorders despite the use of viral vectors such as retroviruses.
In the early steps of infection, retroviruses deliver their nucleoprotein core into the cytoplasm of the target cell. Here, reverse transcription of the viral genome takes place while the core matures into a preintegration complex. The complex must reach the nucleus to achieve integration of the viral DNA into the host cell chromosomes. For simple retroviruses (oncoretroviruses), this step requires the dissolution of the nuclear membrane at mitotic prophase, most likely because the bulky size of the preintegration complex prevents its passive diffusion through the nuclear pores because there are no nuclear localization signals to facilitate active transport into the nucleus.
Currently retroviral vectors used for human gene therapy are replication-defective and must be produced in packaging cells, which contain integrated wild type virus genome sequences and thus provide all of the structural elements necessary to assemble viruses (i.e., the gag, pol, and env gene products), but cannot encapsidate their own wild type virus genomes due to a deletion of the packaging signal sequence (psi).
Replication-defective virus vectors created by removal of the viral structural genes and replacement with therapeutic genes are introduced into the packaging cells; so long as these vectors contain the psi signal, they can take advantage of the structural proteins provided by the cells and be encapsidated into virion. However, after infection of a target cell, the vectors are incapable of secondary horizontal infections of adjacent cells due to the deletion of the essential viral genes.
The use of replication-defective vectors has been an important safeguard against the uncontrolled spread of virus, as replication-competent retroviruses have been shown to cause malignancies in primates (Donahue et al., J. Exp. Med., 1992, 176:1124-1135). However, replication-defective retroviral vectors are produced from the packaging cells at titers on the order of only 106-7 colony-forming units (cfu) per ml, which is barely adequate for transduction in vivo. In fact, clinical trials for gene therapy of glioblastoma multiforme, a highly malignant brain tumor, have encountered major problems in achieving adequate levels of tumor cell transduction, and despite promising initial results in animal studies (Culver et al., Science, 1992, 256:1550-1552). In order to increase transduction levels as much as possible, instead of using a single shot of virus-containing supernatant, the virus packaging cell line PA317 itself was injected into the brain tumors to constitutively produce retrovirus vectors carrying the HSV-tk gene (Oldfield et al., Human Gene Therapy, 1993, 4:39-69). Subsequently, the protocol was further modified to include a debulking procedure followed by multiple injection sites, as it was found that the virus vectors did not diffuse far enough from the site of initial injection. Despite these modifications, the transduction efficiency has been estimated to less than 1% of the tumor cell mass and any significant tumor destruction is presumed to be due to the potent bystander effect of the HSV-tk/ganciclovir treatment. Thus efficient transduction of cancer cells in a solid tumor mass represents a major problem for cancer gene therapy.
Accordingly, there is a need for a gene transfer vector capable of high-level transduction in vivo, while limiting uncontrolled spread of replication-competent virus which could result in insertional mutagenesis and carcinogenesis.
The present invention provides recombinant replication competent retroviral vectors for gene delivery. The vectors provide a high-level of transduction in vivo. The use of replication-competent vectors of the invention allow efficient in vivo transduction. The incorporation of cell-type targeting polynucleotide sequences into such vectors reduce or eliminate the native pathogenic potential of replication-competent retroviruses while improving their target cell specificity.
In one embodiment, the present invention provides a recombinant replication competent retrovirus having a retroviral GAG protein; a retroviral POL protein; a retroviral ENV protein; a retroviral genome comprising Long-Terminal Repeat (LTR) sequences at the 5xe2x80x2 and 3xe2x80x2 ends of the retroviral genome, wherein a target specific polynucleotide sequence is contained within the LTR sequences at the 5xe2x80x2 and/or 3xe2x80x2 end of the retroviral genome, a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and cis-acting nucleic acid sequences, and sequences encoding proteins, necessary for reverse transcription, packaging and integration in a target cell. The target specific polynucleotide sequence of the retroviral vector can be a tissue-specific promoter sequence, for example a sequence associated with a growth regulatory gene, such as, for example, probasin. To target the retrovirus to a specific cell or tissue the retrovirus ENV protein can further comprise a target-specific ligand sequence, which encodes, for example, an antibody, receptor, or ligand, such as, heregulin.
In another embodiment, the present invention provides a recombinant retroviral polynucleotide sequence, having a polynucleotide sequence encoding a GAG protein; a polynucleotide sequence encoding a POL protein; a polynucleotide sequence encoding an ENV protein; a polynucleotide sequence comprising a Long Terminal Repeat (LTR) at the 5xe2x80x2 and 3xe2x80x2 end of the retroviral polynucleotide sequence containing a target specific polynucleotide sequence at the 5xe2x80x2 and or 3xe2x80x2 end; a heterologous polynucleotides sequence operably linked to a regulatory nucleic acid sequence; and cis acting polynucleotide sequence, as well as sequences encoding proteins, necessary for reverse transcription, packaging and integration in a target cell. The target specific polynucleotide sequence is a cell- or tissue-specific promoter sequence such as, for example, one associated with a growth regulatory gene or one associated with a cancer marker (e.g., probasin). The ENV sequence may be further associated with a target-specific ligand polynucleotide sequence, for example a sequence encoding an antibody, a receptor (e.g., a hormone receptor), or a ligand, such as, for example, heregulin.
In yet another embodiment, the present invention provides, a method of treating a subject having a cell proliferative disorder, by contacting the subject with a retrovirus, having a retroviral GAG protein; a retroviral POL protein; a retroviral ENV protein; a retroviral genome comprising Long-Terminal Repeat (LTR) sequences at the 5xe2x80x2 and 3xe2x80x2 end of the retroviral genome, wherein a target specific polynucleotide sequence is contained within the LTR sequences at the 5xe2x80x2 and/or 3xe2x80x2 end of the retroviral genome, a heterologous nucleic acid sequence operably linked to a regulatory nucleic acid sequence; and cis-acting nucleic acid sequences, as well as sequence encoding proteins, necessary for reverse transcription, packaging and integration in a target cell. The target cell is preferably a cell having a cell proliferative disorder, such as a neoplastic cell.
These and other aspects of the present invention will be apparent to those of skill in the art from the teachings herein.